Siliques or pods from Brassica plants release their seeds through a process called fruit dehiscence. A silique consists of two carpels joined margin to margin. The suture between the margins forms a thick rib, called replum. As pod maturity approaches, the two valves separate progressively from the replum, along designated lines of weakness in the pod, eventually resulting in the shattering of the seeds that were attached to the replum. The dehiscence zone defines the exact location of the valve dissociation.
Shedding of seed (also referred to as “seed shatter” or “pod shatter”) by mature pods before or during crop harvest is a universal phenomenon with crops that develop dry dehiscent fruits. Premature seed shatter results in a reduced seed recovery, which represents a problem in crops that are grown primarily for the seeds, such as oil-producing Brassica plants, particularly oilseed rape. Another problem related to premature seed shattering is an increase in volunteer growth in the subsequent crop year. In oilseed rape, pod shatter-related yield losses are on average 20% (Child et al., 1998, J Exp Bot 49: 829-838), but can reach up to 50%, depending on the weather conditions (MacLeod, 1981, Harvesting in Oilseed Rape, pp. 107-120 Cambridge Agricultural Publishing, Cambridge).
Current commercial oilseed rape varieties are extremely susceptible to shattering. There is little variation for resistance to shattering within existing breeding programs of B. napus but resistant lines have been found within the diploid parents of B. napus (B. oleracea and B. rapa) as well as within other members of the Brassica genus, notably B. juncea, B. carinata and B. nigra. Kadkol et al. (1986, Aust. J. Botany 34 (5): 595-601) report increased resistance towards shattering in certain accessions of B. campestris that was associated with the absence of a separation layer in the region of attachment of the siliqua valves to the replum. Prakash and Chopra (1988, Plant breeding 101: 167-168) describe the introgression of resistance to shattering in Brassica napus from Brassica juncea through non-homologous recombination. Spence et al. (1996, J of Microscopy 181: 195-203) describe that some lines of Brassica juncea show a reduced tendency to shatter as compared to Brassica napus lines. Morgan et al., 1998 (Fields Crop Research 58, 153-165) describe genetic variation for pod shatter resistance among lines of oilseed rape developed from synthetic B. napus and conclude that lines which required much energy to open their pods appeared to have increased vascularisation in the dehiscence zone and to have reduced cell wall degradation within the dehiscence zone. They further found a significant negative correlation between the length of the pod beak and the force needed to cause pod shattering. Child and Huttly (1999, Proc 10th Int. Rapeseed Congress) describe variation in pod maturation in an irradiation-induced mutant B. napus and a population of its parent cultivar, Jet Neuf, wherein the most resistant wild-type and mutant plants showed much lignification of groups of cells throughout the dehiscence zone and wherein vascular traces situated close to the inner edge of the dehiscence zone in the mutant were described to help to secure the valves. Child et al. (2003, J Exp Botany 54 (389): 1919-1930) further describe the association between increased pod shatter resistance and changes in the vascular structure in pods of a resynthesized Brassica napus line. However, the traditional methods for breeding have been unsuccessful in introducing shatter resistance into rape cultivars, without interference with other desirable traits such as early flowering, maturity and blackleg resistance (Prakash and Chopra, 1990, Genetical Research 56: 1-2).
Several genes, which promote or inhibit pod dehiscence, have been identified in Arabidopsis thaliana through mutant analysis: Combined mutants in both SHATTERPROOF1 (SHP1; initially referred to as AGL1) and SHATTERPROOF2 (SHP2; initially referred to as AGL5) result in indehiscent siliques (i.e. siliques which remain closed upon maturity in Arabidopsis thaliana) (Liljegren et al., 2000, Nature 404, 766-770). Similarly, mutants in the INDEHISCENT gene (referred to as IND1) in Arabidopsis thaliana (Liljegren et al., 2004, Cell 116: 843-853; PCT publication WO 01/79517), as well as in ALCATRAZ (referred to as ALC; Rajani et al. 2001, Current Biology 11, 1914-1922) interfered with pod dehiscence leading to pod shatter resistance. Constitutive expression of FRUITFUL (FUL), a repressor of SHP and IND, in Arabidopsis thaliana also resulted in indehiscent siliques (Ferrandiz et al., 2000, Science, 289, 436-438). FILAMENTOUS FLOWER (FIL) and YABBY3 (YAB3), two YABBY-family transcription factors (Sawa et al., 1999, Genes Dev 13, 1079-1088; Siegfried et al., 1999, Development 126, 4117-4128), and JAGGED (JAG), a C2H2 zinc-finger transcription factor (Dinneny et al., 2004, Development 131, 1101-1110; Ohno et al., 2004, Development 131, 1111-1122), were identified to redundantly contribute to proper valve and valve margin development by promoting the expression of FUL and SHP in a region-specific manner (Dinneny et al., 2005, Development 132, 4687-4696). Genes for a number of hydrolytic enzymes, such as endopolygalacturonases, which play a role, during pod dehiscence, in the programmed breakdown of the dehiscence zone in pods from Brassica plants have also been identified (see e.g. WO 97/13865; Petersen et al., Plant. Mol. Biol., 1996, 31:517-527).
WO99/00503, WO01/79517 and WO0159122 describe downregulation of the expression of the Arabidopsis ALC, IND, AGL1 and AGL5 genes and orthologs thereof using gene-silencing techniques (such as antisense suppression or cosuppression) and mutagenesis.
WO 2010/006732, describes that the fruit dehiscence properties in Brassica plants can be controlled by controlling the number of IND genes/alleles that are “functionally expressed” in seed pods, i.e. that result in functional (biologically active) IND protein. By combining a number of full knock-out mutant IND alleles, while maintaining a minimal number of wild type IND alleles, resulting in a minimal level of functional IND protein, the dehiscence properties of the seed pods can be modified, more specifically pod shatter resistance can be increased and seed shattering can be reduced, or seed shattering can be delayed until after harvest, while maintaining at the same time an agronomically relevant threshability of the pods, such that the pods may still be opened along the dehiscence zone by applying limited physical forces.
Rajani et al. (2001, Current Biology 11, 1914-1922) describe a recessive mutant in the Arabidopsis ALCATRAZ gene, that disrupts the process of silique dehiscence. ALC encodes a myc/bHLH protein. Both lignification and external appearance of the dehiscence zone remains unchanged in the alc mutant. ALC plays a role in cell separation during fruit dehiscence by promoting the differentiation of a cell layer that is the site of separation between the valves and the replum within the dehiscence zone.
WO2001/059121 and WO2001/059122 also describe an Arabidopsis mutant, SGT10166, having siliques with an indehiscent phenotype. The gene disrupted in this mutant encodes a bHLH protein, and is identical to the ALCATRAZ gene as described by Rajani et al. (2001, Current Biology 11, 1914-1922). Expression of a dominant negative version of the SGT10166 protein (which is identical to the ALCATRAZ protein) delays dehiscence.
Hua et al. (2009, Planta 230: 493-503) cloned and sequenced two ALCATRAZ genes from Brassica napus, BnaC.ALC.a and BnaA.ALC.a. Both genes complement the alc mutation of Arabidopsis thaliana. Southern blot hybridization of Brassica napus ALC genes gave rise to three hybridized bands, indicating multiple copies of the ALC homologs in the genome of Brassica napus. Only expression of BnaC.ALC.a, but not of BnaA.ALC.a was detectable in the silique tissue of Brassica napus. The result indicates that the 5′ flanking sequence of BnaC.ALC.a, not of BnaA.ALC.a could be used to drive antisense or RNAi structures of the gene in the genetic engineering project for anti-pod-shattering agronomic trait. Based on these results, it would be likely that downregulation of BnaA.ALC.a would be sufficient to obtain a podshatter resistant phenotype in Brassica napus. 
It is important to realize that while seed shattering constitutes an important problem in oilseed rape culture, which may be solved by developing pod shatter resistant lines, ultimately, separation of the seeds from the pods is still required. In normal agricultural practice this is achieved by threshing of the pods by a combine harvester. Threshing of the pods by a combine harvester must be complete and must cause minimum damage to the seeds thus released. However, as pod strength increases, the more severe action required to thresh them causes an unacceptable level of damage to the seed. The pods of pod shatter resistant Brassicaceae plants should thus not be so strong that they cannot be threshed in a combine harvester (Bruce et al. 2001, J. Agric. Engng Res. 80, 343-350).
The prior art shows that, in order to obtain podshatter resistance in Brassica, while maintaining agronomically relevant threshability, the extent to which the genes involved in podshatter resistance have to be modulated, may be subtle (WO 2004/113542, WO 2010/006732).
In order to use the ALCATRAZ gene for podshatter resistance while retaining agronomically relevant threshability, a need remains for knowing all ALCATRAZ genes sequences in the Brassica genome. The isolation of mutant alleles corresponding to alc in economically important Brassicaceae plants, such as oilseed rape, is a laborious and time consuming task. Moreover, such isolation may be complicated by the amphidiploidy in oilseed rape and the consequent functional redundancy of the corresponding genes. Although Hua et al. (2009, Planta 230: 493-503) did not detect expression BnaA.ALC.a in the silique tissue of Brassica napus, and thus it is likely that there would be no need to modify BnaA.ALC.a in order to obtain podshatter resistance, a need remains for knowing how, and how many of the Brassica ALCATRAZ genes have to be modified in order to obtain podshatter resistance with agronomically relevant threshability.
These and other objects are achieved by the present invention, as indicated by the various embodiments described in the summary of the invention, figures, detailed description, examples and claims.